SUMMARY The overlying goal of the proposed research program is to understand the molecular mechanisms that control gastrulation using the Drosophila embryo as a model system. In particular, we are interested in investigating how dorsal-ventral (DV) patterning and cell signaling processes are temporally regulated in the early embryo; in deciphering the cis-regulatory mechanisms controlling spatiotemporal gene expression; and studying how collective cell movements are orchestrated. These are inter-related questions that also are relevant for the development of all animals, and as such these studies have the potential to provide far-reaching insights. To assay progression of developmental events, we develop and employ novel technologies for making temporally relevant observations using live in vivo imaging, computation including mathematical modeling, and molecular biology. We have focused on the design and implementation of new imaging approaches that allow us to acquire fine-scale spatiotemporal data of developmental processes, to capture transcription factor dynamics as well as cell movements. Here we propose three research directions to provide further insight into the system of genes driving Drosophila gastrulation. Project 1 involves expansion of DV patterning network with a focus on the regulation of temporal expression. In the early embryo, we have found that transcription factors acting along the DV axis exhibit dynamic changes in levels. Expression profiles for putative target genes at multiple time-points spanning the early development will be obtained at single embryo resolution in wildtype versus mutant embryos to provide insight into how timing of expression is regulated by the gene network. Spatial expression of a subset of genes will be further investigated using in situ hybridization, and live imaging methods will be used to monitor gene expression in real-time. Project 2 will investigate the mechanism and role of coordinate cis-regulatory action using methods to analyze chromatin conformation in vivo within each nucleus of the embryo as well as assays to compare levels and timing of gene expression supported by co-acting cis-regulatory elements. Project 3 will investigate the function and regulation of FGF signaling in migrating cells. We hypothesize that FGF signaling modulates the adhesive properties of mesoderm cells at gastrulation to support their cohesive, organized movement. The role of heparan sulfate proteoglycan molecules and cleavage-state on FGF activity will also be investigated. The overlying goal of the proposed research program is to understand the molecular mechanisms controlling morphogenesis of the embryo through study of a network of genes that controls patterning, signaling pathway activation, and, ultimately, cell movements in the early Drosophila embryo. The conservation of gene regulatory mechanisms across all animals promises that these studies will have far reaching implications. In particular, a better understanding of cis-regulatory mechanisms, in general, has many benefits including improved, targeted gene therapy; while understanding how cell migration is controlled will provide insights toward the regulation of cell metastasis.